Inhibitors of lc3/atg3 interaction and their use in the treatment of cancer

ABSTRACT

The present inventors discovered that the molecule pyridinylthiazolamine (PTA) specifically prevents the protein-protein interaction of hLC3 with hAtg3 in vitro and in cell based assays. The inventors have developed a novel class of PTA analogs which also prevents the protein-protein interaction of hLC3 with hAtg3 in vitro, and in cell based assays, and which can be used in prior to, or in combination with chemotherapeutic agents to treat proliferative diseases such as cancer.

REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/847,593, filed Sep. 8, 2015, now U.S. Pat. No. 10,059,702, issuedAug. 28, 2018, which claims the benefit of Provisional U.S. PatentApplication No. 62/047,271, filed Sep. 8, 2014, the content of each ofthe aforementioned applications is herein incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

Autophagy is a catabolic process performed by eukaryotic cells in orderto maintain homeostasis and to degrade unwanted or toxic cellularcontent such as misfolded proteins. The classical autophagy pathway inmammals is depicted in FIG. 1A. Observations indicate a critical role ofautophagy in cancer cells e.g. survival of colorectal tumors isabolished when treated with autophagy inhibitors. However, the exactrole of autophagy in cancer is very complex and highly debated. On theone hand, autophagy has been associated with increased survival oftumors, for example, under hypoxic or nutrient deprivation conditions;on the other hand, autophagy prevents DNA damage in normal cells, andtherefore can serve a tumor suppressing role within the cell.

Many autophagy inhibitors such as 3-methyladenine (3-MA) orhydroxychloroquine (CQ) are promiscuous compounds. Moreover, manyFDA-approved drugs interact with multiple protein targets. Inhibition ofmTOR through rapamycin leads to an activation of autophagy, whileinhibition of VPS34 by Wortmannin prevents autophagy (FIG. 1A). Inaddition, the protein targets mTOR or Beclin1 are effectors of multiplepathways and are therefore pleiotropic by nature.

Current intervention methods targeting the autophagy pathway aresomewhat indirect and intervene very early in the pathway, which mayallow the cell to regulate the autophagy pathway through othercheckpoint events. Recently, a research group at Sanofi recognized thelack of direct autophagy inhibitors and its negative impact on thecancer field. They utilized an assay comprising a fusion constructexpressing enhanced green fluorescent protein (EGFP)-LC3 in two tumorcell lines and monitored the granular fluorescence in the presence ofsmall molecules. Hence, they were able to directly measure the effect ofthe small molecules on the lipidation state of EGFP-LC3, using loss ofgranulosity as an indicator of the unlipidated form. This assay wasfollowed with a second assay which looked specifically at the inductionof cell death under starvation conditions in the presence of the smallmolecules identified in the primary screen. Both can be used to screennovel compounds for inhibition of LC3/Atg3 interaction.

As such, there still exists an unmet need for novel compounds which canact as direct autophagy inhibitors and their use in the study andtreatment of cancer.

SUMMARY OF THE INVENTION

The present inventors discovered that PTA specifically prevents theprotein-protein interaction of hLC3 with hAtg3 in vitro. The inventorshave created a novel class of PTA analogs which also prevents theprotein-protein interaction of hLC3 with hAtg3 in cell cultures, andwhich can be used to treat proliferative diseases such as cancer.

In accordance with an embodiment, the present invention provides acompound of formula I:

or salt solvate, or stereoisomer thereof, wherein R₁ is an aryl,pyrimidyl, napthalenyl or heteronapthalenyl group which may besubstituted with one or more C₁-C₆ alkyl or substituted groups; whereinL is a linker group of 0 or 1, comprising an alkylamino group; whereinR₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄ with oneor more C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylamino C₁-C₆ alkyl,C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ alkylthio C₁-C₆ alkyl, C₆-C₁₄arylthio, C₁-C₆ alkylsulfonyl C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₃-C₈ cycloalkyl, heterocyclyl, C₁-C₆alkylamino, di C₁-C₆ alkylamino, C₁-C₆ alkylthio, C₂-C₆ alkenylthio,C₂-C₆ alkynylthio, C₆-C₁₄ aryloxy, C₂-C₆ acyloxy, thio C₂-C₆ acyl,amido, and sulphonamido, and C₁-C₆ alkyl, and C₂-C₆ alkenyl, C₂-C₆alkynyl, or a halogen, wherein each of alkyl, aryl, or heterocyclylmoiety may be unsubstituted or substituted with one or more substituentsselected from the group consisting of halo, hydroxy, carboxy,phosphoryl, phosphonyl, phosphono C₁-C₆ alkyl, carboxy C₁-C₆ alkyl,dicarboxy C₁-C₆ alkyl, dicarboxy halo C₁-C₆ alkyl, sulfonyl, cyano,nitro, alkoxy, alkylthio, acyl, acyloxy, thioacyl, acylthio, aryloxy,amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino,guanidino, aldehydo, ureido, and aminocarbonyl.

In accordance with an embodiment, the present invention providescompounds of formula II:

wherein R₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄with one or more C₁-C₆ alkyl groups or a halogen, and R₃ is one or moreC₁-C₆ alkyl or substituted groups.

In accordance with an embodiment, the present invention provides acompound of formula III:

wherein R₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄with one or more C₁-C₆ alkyl groups or a halogen, and R₃ is one or moreC₁-C₆ alkyl or substituted groups.

In accordance with an embodiment, the present invention provides acompound of formula IV:

wherein R₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄with one or more C₁-C₆ alkyl groups or a halogen, and R₃ is one or moreC₁-C₆ alkyl or substituted groups.

In accordance with another embodiment, the present invention providescompounds of formula I, wherein the compound is selected from the groupconsisting of:

In accordance with a further embodiment, the present invention providesa pharmaceutical composition comprising one or more compounds of formulaI, as described above, or a salt, solvate or stereoisomer thereof, and apharmaceutically acceptable carrier.

In accordance with another embodiment, the present invention provides apharmaceutical composition one or more compounds of formula I, asdescribed above, or a salt, solvate or stereoisomer thereof, and apharmaceutically acceptable carrier and further comprises at least onechemotherapeutic agent.

In accordance with another embodiment, the present invention provides apharmaceutical composition as described above, for use in a medicament,preferably for use in a medicament for the treatment of cancer or aproliferative disease in a subject comprising administering to thesubject an effective amount of the medicament.

In accordance with another embodiment, the present invention provides amethod for treating cancer in a subject comprising administering to thesubject an effective amount of a compound of formula I, or a saltsolvate, or stereoisomer thereof.

In accordance with a further embodiment, the present invention providesa method for treating cancer in a subject comprising administering tothe subject an effective amount of a pharmaceutical compositioncomprising a compound of formula I, or a salt solvate, or stereoisomerthereof, and a pharmaceutically effective carrier.

In accordance with yet another embodiment, the present inventionprovides a method for treating cancer in a subject comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition comprising a compound of formula I, or a salt solvate, orstereoisomer thereof, at least one chemotherapeutic agent, and apharmaceutically effective carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are an overview of the mammalian autophagy pathway. 1A)Proteins essential for autophagy are depicted as rectangles. Ovalsrepresent proteins whose essentiality is not known. Indicated in red aretwo known inhibitors used in cancer treatment. Purple signifies theinteraction targeted by our PTA molecule. Figure adapted from Hain etal. 2013. 1B) Schematic of the proteins involved in the LC3/Atg8PE-conjugation pathway.

FIGS. 2A-2C depict the properties of the PTA-binding site on hLC3. 2A)Energy-minimized docking pose of PTA to hLC3. The pyridinylthiazolaminering system predominantly binds to the W-site of hLC3. 2B) Conservationof amino acids between hLC3 and Plasmodium Atg8. Shown in yellow sticksis the bound peptide of Atg3. The W-Site is highly conserved, whereasthe L-site is less conserved, explaining the lower affinity of hLC3 toPTA (FIGS. 2A, 2B adapted from Hain et al. 2013). 2C) Direct bindinginteraction of purified hLC3 or PfAtg8 to PTA immobilized on a SPR chipto determine their respective affinities.

FIG. 3 shows the conservation of the W-/L-site and their respectivebinding affinity to PTA. To maintain the identical view, residuedifferences were painted onto the PfAtg8 structure. Residues in theproximity of the W-/L-site of hLC3 lead to lower binding affinities ofour current molecule targeting the Atg8-Atg3 interaction.

FIG. 4 provides an anti-LC3 Western blot of PTA treated HCT8 coloncancer cells with corresponding Coomassie stained 4-20% SDS-PAGE. Thearrows indicate the band used for quantification with ImageJ. The SDSgel indicates approximate equal amounts were loaded per lane.

FIG. 5 depicts the concentration dependent effect of PTA and asynthesized derivative (Compound 10) on two cancer cell lines in a HUVECimpedance assay. PTA or Compound 10 were added ranging from 100 μM to1.5 μM to the cells after 12 hours. The apparent IC₅₀ after 72 hours ofdrug treatment was calculated and is shown in the inset of each graph ofthe triplicate impedance measurement per concentration. The DMSO vehiclecontrol is shown as dark red line. The observed IC₅₀ for PTA treatmentin cells are in the same range as the affinity of hLC3 to PTA on the SPRchip of 18 μM.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors recently identified anti-malarial compounds with aPyridinylthiazolamine (PTA) scaffold inhibit the criticalphosphatidylethanolamine (PE)-conjugation step of LC3/Atg8 (FIG. 1) (J.Med. Chem. 2014, 57, 4521-4531 (2014)), included in U.S. PatentApplication No. 61/984,315, and incorporated by reference herein. Theinventors' PTA compounds were designed to interfere with the Atg8-Atg3protein-protein interaction and thereby prevent the lipidation of Atg8(hLC3-II). Only the PE-conjugated form (hLC3-II) participates inelongation of the autophagosomal membrane, preparing the encapsulatedmaterial for fusion with the lysosome for nutrient recycling anddegradation processes. Before conjugation to PE, most LC3/Atg8orthologues require proteolytic processing of one or several amino acidsby Atg4 to expose a C-terminal glycine (FIG. 1B). The exposed C-terminusof Atg8 is then attached to its E1 activating enzyme, Atg7, through athioester bond that requires activation by ATP hydrolysis. Atg8 is thentransferred to its E2 conjugating enzyme, Atg3, to form anotherthioester bond before being transferred to PE in the incipientphagophore membrane. This complex activation cascade yielding LC3-II canbe regulated at any of the above steps. Therefore inhibiting the verylast step in this conjugation process can be a very effective approach,as no feedback to the prior activation steps is possible.

In accordance with an embodiment, the present invention provides acompound of formula I:

or salt solvate, or stereoisomer thereof, wherein R₁ is an aryl,pyrimidyl, napthalenyl or heteronapthalenyl group which may besubstituted with one or more C₁-C₆ alkyl or substituted groups; whereinL is a linker group of 0 or 1, comprising an alkylamino group; whereinR₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄ with oneor more C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylamino C₁-C₆ alkyl,C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ alkylthio C₁-C₆ alkyl, C₆-C₁₄arylthio, C₁-C₆ alkylsulfonyl C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₃-C₈ cycloalkyl, heterocyclyl, C₁-C₆alkylamino, di C₁-C₆ alkylamino, C₁-C₆ alkylthio, C₂-C₆ alkenylthio,C₂-C₆ alkynylthio, C₆-C₁₄ aryloxy, C₂-C₆ acyloxy, thio C₂-C₆ acyl,amido, and sulphonamido, and C₁-C₆ alkyl, and C₂-C₆ alkenyl, C₂-C₆alkynyl, or a halogen, wherein each of alkyl, aryl, or heterocyclylmoiety may be unsubstituted or substituted with one or more substituentsselected from the group consisting of halo, hydroxy, carboxy,phosphoryl, phosphonyl, phosphono C₁-C₆ alkyl, carboxy C₁-C₆ alkyl,dicarboxy C₁-C₆ alkyl, dicarboxy halo C₁-C₆ alkyl, sulfonyl, cyano,nitro, alkoxy, alkylthio, acyl, acyloxy, thioacyl, acylthio, aryloxy,amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino,guanidino, aldehydo, ureido, and aminocarbonyl.

In the compounds disclosed herein, including, e.g., General Formula I,the “hydrocarbon group” of the “hydrocarbon group which may besubstituted” represented by R₁ may be exemplified by a straight-chainedor cyclic hydrocarbon group (e.g., an alkyl group, an alkenyl group, analkynyl group, a cycloalkyl group, an aryl group, an arylalkyl group,alkylol group, hydroxyalkyl group, alkoxy group, alkoxyalkyl group, abranched or straight-chain alkylamino, dialkylamino, or alkyl ordialkylaminoalkyl, or thioalkyl, thioalkenyl, thioalkynyl, aryloxy,thioaryl, thioheteroaryl, acyloxy, thioacyl, amido, sulphonamido, etc.),or the like. Among these, straight-chained or cyclic hydrocarbon groupshaving 1 to 16 carbon atoms are preferred.

Examples of the “cycloalkyl group” preferably include a C₃₋₈ cycloalkylgroup (e.g., a cyclopropyl group, a cyclobutyl group, a cyclopentylgroup, a cyclohexyl group, etc.) and the like.

Examples of the “aryl group” preferably include a C₆₋₁₄ aryl group(e.g., a phenyl group, a 1-naphthyl group, a 2-naphthyl group and thelike.

Examples of the “arylalkyl group” preferably include a C₆₋₁₄ arylalkylgroup (e.g., a benzyl group, a phenylethyl group, a diphenylmethylgroup, a 1-naphthylmethyl group, a 2-naphthylmethyl group, a2,2-diphenylethyl group, a 3-phenylpropyl group, a 4-phenylbutyl group,a 5-phenylpentyl group, etc.) and the like.

The term “hydroxyalkyl” embraces linear or branched alkyl groups havingone to about ten carbon atoms any one of which may be substituted withone or more hydroxyl groups.

The term “alkylamino” includes monoalkylamino. The term “monoalkylamino”means an amino, which is substituted with an alkyl as defined herein.Examples of monoalkylamino substituents include, but are not limited to,methylamino, ethylamino, isopropylamino, t-butylamino, and the like. Theterm “dialkylamino” means an amino, which is substituted with two alkylsas defined herein, which alkyls can be the same or different. Examplesof dialkylamino substituents include dimethylamino, diethylamino,ethylisopropylamino, diisopropylamino, dibutylamino, and the like.

The terms “alkylthio,” “alkenylthio” and “alkynylthio” group mean agroup consisting of a sulphur atom bonded to an alkyl-, alkenyl- oralkynyl- group, which is bonded via the sulphur atom to the entity towhich the group is bonded.

In accordance with an embodiment, the present invention providescompounds of formula II:

wherein R₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄with one or more C₁-C₆ alkyl groups or a halogen, and R₃ is one or moreC₁-C₆ alkyl or substituted groups.

In accordance with an embodiment, the present invention provides acompound of formula III:

wherein R₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄with one or more C₁-C₆ alkyl groups or a halogen, and R₃ is one or moreC₁-C₆ alkyl or substituted groups.

In accordance with an embodiment, the present invention provides acompound of formula IV:

wherein R₂ is aryl or pyrimidyl group which may be substituted at C₂-C₄with one or more C₁-C₆ alkyl groups or a halogen, and R₃ is one or moreC₁-C₆ alkyl or substituted groups.

In accordance with another embodiment, the present invention providescompounds of formula I, wherein the compound is selected from the groupconsisting of:

In accordance with an embodiment, the present invention provides apharmaceutical composition comprising the compounds of formula I-IV, ora salt, solvate, stereoisomer, or prodrug thereof, and apharmaceutically acceptable carrier.

Included within the compounds of the present invention are thetautomeric forms of the disclosed compounds, isomeric forms includingdiastereoisomers, and the pharmaceutically-acceptable salts thereof. Theterm “pharmaceutically acceptable salts” embraces salts commonly used toform alkali metal salts and to form addition salts of free acids or freebases. Examples of acids which may be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulphuric acid and phosphoric acid, and such organicacids as maleic acid, succinic acid and citric acid. Otherpharmaceutically acceptable salts include salts with alkali metals oralkaline earth metals, such as sodium, potassium, calcium and magnesium,or with organic bases, such as dicyclohexylamine. Suitablepharmaceutically acceptable salts of the compounds of the presentinvention include, for example, acid addition salts which may, forexample, be formed by mixing a solution of the compound according to theinvention with a solution of a pharmaceutically acceptable acid, such ashydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid,maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid,citric acid, tartaric acid, carbonic acid or phosphoric acid. All ofthese salts may be prepared by conventional means by reacting, forexample, the appropriate acid or base with the corresponding compoundsof the present invention.

Salts formed from free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

For use in medicines, the salts of the compounds of the presentinvention should be pharmaceutically acceptable salts. Other salts may,however, be useful in the preparation of the compounds according to theinvention or of their pharmaceutically acceptable salts.

In accordance with another embodiment, the present invention provides apharmaceutical composition as described above, for use in a medicament,preferably for use in a medicament for the treatment of cancer or aproliferative disease in a subject comprising administering to thesubject an effective amount of the medicament.

In addition, embodiments of the invention include hydrates of thecompounds of the present invention. The term “hydrate” includes but isnot limited to hemihydrate, monohydrate, dihydrate, trihydrate and thelike. Hydrates of the compounds of the present invention may be preparedby contacting the compounds with water under suitable conditions toproduce the hydrate of choice.

Thus, in accordance with an embodiment, the present invention providesthe compounds of formulas I-IV, or a pharmaceutical compositioncomprising the compounds of formulas I-IV, as described herein, as aninhibitor of LC3-Atg3 interaction in a subject.

In accordance with another embodiment, the present invention providesthe compounds of formulas I-IV, or a pharmaceutical compositioncomprising the compounds of formulas I-IV, as described herein, as aninhibitor of hLC3-hAtg3 interaction in a subject.

In accordance with a further embodiment, the present invention providesthe compounds of formulas I-IV, or a pharmaceutical compositioncomprising the compounds of formulas I-IV, as described herein, and atleast one chemotherapeutic agent for treating a hyperproliferativedisease or cancer in a subject.

Embodiments of the invention include a process for preparingpharmaceutical products comprising the compounds, salts, solvates orstereoisomers thereof. The term “pharmaceutical product” means acomposition suitable for pharmaceutical use (pharmaceuticalcomposition), as defined herein.

As used herein, the term “treat,” as well as words stemming therefrom,includes preventative as well as disorder remitative treatment. Theterms “reduce”, “suppress” and “inhibit,” as well as words stemmingtherefrom, have their commonly understood meaning of lessening ordecreasing. These words do not necessarily imply 100% or completetreatment, reduction, suppression, or inhibition.

As used herein, the term “subject” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Lagomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

With respect to pharmaceutical compositions described herein, thepharmaceutically acceptable carrier can be any of those conventionallyused, and is limited only by physico-chemical considerations, such assolubility and lack of reactivity with the active compound(s), and bythe route of administration. The pharmaceutically acceptable carriersdescribed herein, for example, vehicles, adjuvants, excipients, anddiluents, are well-known to those skilled in the art and are readilyavailable to the public. It is preferred that the pharmaceuticallyacceptable carrier be one which is chemically inert to the activeagent(s), and one which has little or no detrimental side effects ortoxicity under the conditions of use. Examples of the pharmaceuticallyacceptable carriers include soluble carriers such as known buffers whichcan be physiologically acceptable (e.g., phosphate buffer) as well assolid compositions such as solid-state carriers or latex beads.

The carriers or diluents used herein may be solid carriers or diluentsfor solid formulations, liquid carriers or diluents for liquidformulations, or mixtures thereof.

Solid carriers or diluents include, but are not limited to, gums,starches (e.g., corn starch, pregelatinized starch), sugars (e.g.,lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g.,microcrystalline cellulose), acrylates (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may be,for example, aqueous or non-aqueous solutions, suspensions, emulsions oroils. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, and injectable organic esters such as ethyl oleate.Aqueous carriers include, for example, water, alcoholic/aqueoussolutions, cyclodextrins, emulsions or suspensions, including saline andbuffered media.

Examples of oils are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, mineral oil, olive oil,sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil,olive, petrolatum, and mineral. Suitable fatty acids for use inparenteral formulations include, for example, oleic acid, stearic acid,and isostearic acid. Ethyl oleate and isopropyl myristate are examplesof suitable fatty acid esters.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include, for example, sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's andfixed oils. Formulations suitable for parenteral administration include,for example, aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain anti-oxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives.

Intravenous vehicles include, for example, fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Examples are sterile liquids such as water andoils, with or without the addition of a surfactant and otherpharmaceutically acceptable adjuvants. In general, water, saline,aqueous dextrose and related sugar solutions, and glycols such aspropylene glycols or polyethylene glycol are preferred liquid carriers,particularly for injectable solutions.

In addition, in an embodiment, the compounds of the present inventionmay further comprise, for example, binders (e.g., acacia, cornstarch,gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g.,cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelosesodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g.,Tris-HCl, acetate, phosphate) of various pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., cremophor, glycerol,polyethylene glycerol, benzlkonium chloride, benzyl benzoate,cyclodextrins, sorbitan esters, stearic acids), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite, butylated hydroxyanisole),stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethylcellulose), viscosity increasing agents (e.g., carbomer, colloidalsilicon dioxide, ethyl cellulose, guar gum), sweetners (e.g., aspartame,citric acid), preservatives (e.g., thimerosal, benzyl alcohol,parabens), lubricants (e.g., stearic acid, magnesium stearate,polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidalsilicon dioxide), plasticizers (e.g., diethyl phthalate, triethylcitrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodiumlauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines),coating and film forming agents (e.g., ethyl cellulose, acrylates,polymethacrylates), and/or adjuvants.

The choice of carrier will be determined, in part, by the particularcompound, as well as by the particular method used to administer thecompound. Accordingly, there are a variety of suitable formulations ofthe pharmaceutical composition of the invention. The followingformulations for parenteral, subcutaneous, intravenous, intramuscular,intraarterial, intrathecal and interperitoneal administration areexemplary, and are in no way limiting. More than one route can be usedto administer the compounds of the present invention, and in certaininstances, a particular route can provide a more immediate and moreeffective response than another route.

Suitable soaps for use in parenteral formulations include, for example,fatty alkali metal, ammonium, and triethanolamine salts, and suitabledetergents include, for example, (a) cationic detergents such as, forexample, dimethyl dialkyl ammonium halides, and alkyl pyridiniumhalides, (b) anionic detergents such as, for example, alkyl, aryl, andolefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, andsulfosuccinates, (c) nonionic detergents such as, for example, fattyamine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylenecopolymers, (d) amphoteric detergents such as, for example,alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammoniumsalts, and (e) mixtures thereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the compound of the present invention or a salt,solvate or stereoisomer thereof, in solution. Preservatives and buffersmay be used. In order to minimize or eliminate irritation at the site ofinjection, such compositions may contain one or more nonionicsurfactants, for example, having a hydrophile-lipophile balance (HLB) offrom about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include, for example, polyethylene glycol sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol.

The parenteral formulations can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630(2009)).

For purposes of the invention, the amount or dose of the compound of thepresent invention, or a salt, solvate or stereoisomer thereof,administered should be sufficient to effect, e.g., a therapeutic orprophylactic response, in the subject over a reasonable time frame. Thedose will be determined by the efficacy of the particular compound andthe condition of a human, as well as the body weight of a human to betreated.

The dose of the compounds of formulas I-IV of the present invention, ora salt, solvate or stereoisomer thereof, also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular compound. Typically, anattending physician will decide the dosage of the compound with which totreat each individual patient, taking into consideration a variety offactors, such as age, body weight, general health, diet, sex, compoundto be administered, route of administration, and the severity of thecondition being treated. By way of example, and not intending to limitthe invention, the dose of the compound can be about 0.001 to about 100mg/kg body weight of the subject being treated/day, preferably about 1mg/kg/day to about 50 mg/kg/day. In some embodiments, the dosage levelsof the compounds would be in the range of 10 μM to about 500 μM,preferably about 100 μM to about 300 μM.

Alternatively, the compounds of formulas I-IV of the present invention,or a salt, solvate or stereoisomer thereof, can be modified into a depotform, such that the manner in which the compound is released into thebody to which it is administered is controlled with respect to time andlocation within the body (see, for example, U.S. Pat. No. 4,450,150).Depot forms of compound can be, for example, an implantable compositioncomprising the compound and a porous or non-porous material, such as apolymer, wherein compound is encapsulated by or diffused throughout thematerial and/or degradation of the non-porous material. The depot isthen implanted into the desired location within the body and thecompounds are released from the implant at a predetermined rate.

In one embodiment, the compounds of formulas I-IV of the presentinvention, or salts, solvates or stereoisomers thereof, provided hereincan be controlled release compositions, i.e., compositions in which theone or more compounds are released over a period of time afteradministration. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Inanother embodiment the composition is an immediate release composition,i.e., a composition in which all or substantially all of the compoundsof formulas I—IV is released immediately after administration.

In yet another embodiment, the compounds of the present invention can bedelivered in a controlled release system. For example, the agent may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, or other modes of administration. In an embodiment, apump may be used. In one embodiment, polymeric materials can be used. Inyet another embodiment, a controlled release system can be placed inproximity to the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Design of ControlledRelease Drug Delivery Systems, Xiaoling Li and Bhaskara R. Jasti eds.(McGraw-Hill, 2006)).

The compounds of the present invention, or salts, solvates orstereoisomers thereof, may also include incorporation of the activeingredients into or onto particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, hydrogels, etc., or ontoliposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance.

In accordance with the present invention, the compounds may be modifiedby, for example, the covalent attachment of water-soluble polymers suchas polyethylene glycol, copolymers of polyethylene glycol andpolypropylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinylpyrrolidone or polyproline. The modified compounds areknown to exhibit substantially longer half-lives in blood followingintravenous injection, than do the corresponding unmodified compounds.Such modifications may also increase the compounds' solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound abducts less frequently, or in lower doses thanwith the unmodified compound.

It will be understood by those of ordinary skill in the art that thecompounds described above can act as a “chemosensitizing” agent,allowing cancer cells or tumor cells to become more susceptible to theactions of one or more other chemotherapeutic agents, thereby increasingthe chemotherapeutic effect and/or allowing a lower dosage of thechemotherapeutic agents to have a therapeutic effect on the tumor orcancer cells.

As used herein, the term “proliferative disease” includes cancer andother diseases such as neoplasias and hyperplasias. Cellularproliferative diseases include, for example, rheumatoid arthritis,inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas,lipomas, hemangiomas, fibromas, vascular occlusion, restenosis,artherosclerosis, a pre-neoplastic lesion, carcinoma in situ, oral hairyleukoplakia, or psoriasis.

In accordance with one or more embodiments, the term “cancer” caninclude any cancer, including any of acute lymphocytic cancer, acutemyeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer,breast cancer, cancer of the anus, anal canal, or anorectum, cancer ofthe eye, cancer of the intrahepatic bile duct, cancer of the joints,cancer of the neck, gallbladder, or pleura, cancer of the nose, nasalcavity, or middle ear, cancer of the oral cavity, cancer of the vulva,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor.Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer,liver cancer, lung cancer, malignant mesothelioma, melanoma, multiplemyeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cellcarcinoma (RCC)), small intestine cancer, soft tissue cancer, stomachcancer, testicular cancer, thyroid cancer, cancers of the brain,including, for example, gliomas and neuroblastomas, ureter cancer, andurinary bladder cancer.

In an embodiment, the term “administering” means that the compounds ofthe present invention are introduced into a subject, preferably asubject receiving treatment for a proliferative disease, and thecompounds are allowed to come in contact with the one or more diseaserelated cells or population of cells in vivo.

It will be understood by those of ordinary skill in the art that theterm “tumor cell” as used herein means a neoplastic growth which may, ormay not be malignant. Additionally, the compositions and methodsprovided herein are not only useful in the treatment of tumors, but intheir micrometastses and their macrometastses. Typically,micrometastasis is a form of metastasis (the spread of a cancer from itsoriginal location to other sites in the body) in which the newly formedtumors are identified only by histologic examination; micrometastasesare detectable by neither physical exam nor imaging techniques. Incontrast, macrometastses are usually large secondary tumors.

In accordance with an embodiment, the present invention providescompositions and methods for the prevention and/or treatment of tumors,and their micrometastses and their macrometastses.

The term “therapeutic agent” or “chemotherapeutic agent” as well aswords stemming therefrom, as used herein, generally includespharmaceutically or therapeutically active compounds that work byinterfering with DNA synthesis or function in cancer cells. Based ontheir chemical action at a cellular level, chemotherapeutic agents canbe classified as cell-cycle specific agents (effective during certainphases of cell cycle) and cell-cycle nonspecific agents (effectiveduring all phases of cell cycle). Without being limited to anyparticular example, examples of chemotherapeutic agents can includealkylating agents, angiogenesis inhibitors, aromatase inhibitors,antimetabolites, anthracyclines, antitumor antibiotics, monoclonalantibodies, platinums, topoisomerase inhibitors, and plant alkaloids.

In a further embodiment, the compositions and methods of the presentinvention can be used in combination with one or more additionaltherapeutically active agents which are known to be capable of treatingconditions or diseases discussed above. For example, the compositions ofthe present invention could be used in combination with one or moreknown therapeutically active agents, to treat a proliferative disease.Non-limiting examples of other therapeutically active agents that can bereadily combined in a pharmaceutical composition with the compositionsand methods of the present invention are enzymatic nucleic acidmolecules, allosteric nucleic acid molecules, antisense, decoy, oraptamer nucleic acid molecules, antibodies such as monoclonalantibodies, small molecules, and other organic and/or inorganiccompounds including metals, salts and ions.

EXAMPLES

To be functional in autophagy processes, hLC3 undergoes two criticalactivation processes. First a C-terminal glycine has to be exposedthrough proteolytic cleavage by the serine protease Atg4. Second, alipid, phosphatidylethanolamine (PE), is conjugated to the C-terminalglycine residue resulting in hLC3-PE (hLC3-II). The inventors previouslydiscovered 4-formyl-N-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide (PTA)as an autophagy inhibitor in their studies on the plasmodial system insearch of protein-protein interaction inhibitors of the PfAtg8-PfAtg3interaction, the homologous system in the malaria parasite. Thelipidated form of either Atg8 or hLC3 (hLC3-II) can be resolved from theunlipidated form (hLC3-I) using SDS-PAGE due to their differentmigration speeds. Specific commercial antibodies for hLC3 detection wereused in these studies to quantify the two species via Western blot intreated and untreated cancer cell lines.

MCF-7 cells were maintained in RPMI media supplemented with 10% fetalbovine serum. HepG2 cells were maintained in Dulbecco's modified Eaglemedium (DMEM, Invitrogen) media supplemented with 10% fetal bovine serumand 1×MEM non-essential amino acids. For cytotoxicity experiments, cellswere plated on T-75 flasks and grown until they reached at 70-80%confluency.

Cell proliferation was monitored in real-time cell electronic sensing(xCELLigence System, Acea Biosciences) through gold microelectrodearrays on the bottom of 96 E-Plates. 100 μl of corresponding culturemedia was added into each well of 96 E-Plates, which inserted into thestation and background measurement was taken. Then, cells (5.000cells/well in 100 μl) were seeded into 96 E-Plates containing 100 μlmedium/well. After about 20 hours, 200 μl medium was discarded andreplaced with 100 μl of fresh medium containing indicated amounts ofcompounds for each well. In the xCELLigence System the changes in cellnumber are detected as modifications in the measurement of electricalimpedance and are represented as Cell Index (CI).

Example 1

Computational Docking Studies.

Computational docking studies were performed to assess the binding siteof PTA to hLC3 (FIG. 2A) using the OpenEye docking software FRED(OpenEye Scientific Software Inc., Santa Fe, N. Mex.). The highestscoring pose indicates binding of PTA predominantly to the W-/L-site.The L-site in the hLC3 crystal structure does not provide a deep grooveas is the case in the Plasmodium structure described previously, butrather a more extended, flat hydrophobic patch.

Example 2

Surface Plasmon Resonance Analysis.

In comparing the sequence conservation of this W-/L-site between PfAtg8and hLC3, it is apparent that the W-site has maintained a higher degreeof conservation, as indicated by the magenta patch in FIG. 2B. To testwhether or not the W-/L-site of Atg8 was indeed the interaction area towhich our small molecules bind, we performed SPR binding studies withdifferent Atg8-homologs, essentially mimicking mutational studies in theenvironment of the W-/L-site. This allowed us to explore the keyfeatures of the binding site in a manner that was much more efficientthan introducing multiple single point mutations for each protein ofinterest at a time.

SPR studies were performed on PfAtg8 and hLC3 binding to the PTAmolecule immobilized on an SPR chip. The results of these studies areconsistent with observations about the mutations present in therespective W-/L-binding site (FIG. 3), confirming the computationaldocking studies and increasing the confidence in the proposed model forPTA binding. The color coding in FIG. 3 highlights divergent amino acidresidues in red, which is well reflected in the decrease of bindingaffinity for hLC3 versus PfAtg8 to the immobilized PTA-molecule on theSPR chip.

Example 3

PTA-Susceptibility Screen of Cancer Cell Lines.

To show that PTA-molecules are able to bind to hLC3 in cell cultures,and prevent the lipidation of hLC3-I to hLC3-II, we exposed a humancolon cancer cell line (HCT8) to PTA with concentrations ranging fromabout 100 μM to 6.25 μM. We used a DMSO-vehicle control. All experimentswere carried out at 1% final DMSO concentration for 24 hours at 37° C.and 5% CO₂. Visual inspection by microscopy revealed attached and normallooking cells in the DMSO control only, and it revealed detached,blebbing cells with a concentration dependent phenotype in all of thetreated samples. Only at the lowest concentration of 6.75 μM PTA were afew patches of attached cells still visible. The DMSO control sample wastrypsinized to detach the cells from the culture dish. All cells werecollected and spun down before resuspending them in SDS-loading dye forSDS-PAGE and Western blot analysis. Commercial monoclonal antibodiesdirected against hLC3 (D3U4C Rabbit mAB) and GAPDH (14C10 Rabbit mAB) asloading control were used. The Western blot clearly distinguishesbetween the un-lipidated (hLC3-I) and lipidated (hLC3-II) form by theirmigration speed in the SDS-gel (FIG. 4). The lipidated form migratesfaster in an SDS-gel due to its additional negative charge from theconjugated phospholipid. An accumulation of the slower migrating hLC3-Iband is visible, indicating that the PTA molecule of the presentinvention is capable of disrupting the lipidation stage in human cancercell lines.

A concentration of 30 μM PTA for 96 hours was well tolerated in HC-04liver cells, without exhibiting a cytotoxic effect. The same assay willthen be applied to our collection of cancer cell lines to assess whichof the lines exhibit a similar decrease of the population of LC3-II anddetermine the respective IC₅₀ in a first-pass 6-point dose-dependentassay ranging from 20 μM to 80 nM. 0.5% DMSO will be used as a vehiclecontrol and 33 nM Wortmannin as a positive control for autophagyinhibition.

Example 4

Our structural understanding of how the hLC3 binds the PTA-scaffoldcombined with surface plasmon resonance (SPR)-interaction studies, hasenabled the inventors to design the PTA-analogs of the present inventionwith low K_(off)-rates. A low K_(off)-rate indicates that the ligand isbound to the target protein for a longer period of time as compared to aligand with a fast K_(off)-rate. These new PTA-analogs are tested inSPR-based assays with purified recombinant hLC3 and hAtg3 to assesstheir ability to prevent the protein-protein interaction. Newly emergingmolecules from our efforts are subjected to dose-dependent cell basedscreening with our cancer line collection.

Knowledge of the exact properties of the residues surrounding thebinding pocket of hLC3 allows for the generation of specific, rationallydesigned probes that fit snugly into this pocket. The gold standard forthis type of procedure is to generate these designs in correspondencewith crystal structure, because the chemical properties and environmentof the pocket can be defined and complemented by the results ofmutagenesis and functional studies. Understanding the binding mode ofthe substrates Atg3 and Atg7 to hLC3 is important, since this knowledgewill help to suggest properties that are likely to be required for smallmolecule probes to inhibit hLC3-PE conjugation at two differentinterfaces. Our co-crystal structure of the plasmodial PfAtg8 withPfAtg3 peptide has provided important insights, leading to thedevelopment of the first inhibitory molecules directly targeting thisprotein interaction.

About 120 PTA-homologs were identified in the PubChem database(pubchem.ncbi.nlm.nih.gov) that are available through commercialvendors. In a first assessment to determine which homologs to focus oninitially, virtual library screening was performed with the known hLC3structures (1UGM, 3ECI, 3WAL, 3WAO). Comparing the docking results fromdifferent structures of hLC3 allowed us to assemble a short list of ninehigh priority compounds (compounds 1-9). Further small molecules withthe pyridinylthiazolamine (PTA) ring system will be identified, whichdemonstrate interaction with Atg8/LC3 and to improve the kineticparameters of the small molecule binding with its target. Twocomplementary assays will be utilized, the first assesses the smallmolecule's ability to block the protein-protein interaction of hLC3 withhAtg3. The second assay is used to directly measure the binding of thesmall molecule to immobilized hLC3 on an SPR chip, which will allow usto draw conclusions about the physical interaction between the twobinding partners.

Example 5

To define the range of metastasizing cancer cell lines susceptible toPTA derivatives of the present invention, we can use the HUVECelectrical-impedance assay. Invasive tumor cell lines are monitoredthrough electric impedance measurements of human umbilical veinendothelial cells (HUVEC) monolayers in the presence of PTA derivatives.In this assay a monolayer of human umbilical vein endothelial cells(HUVEC) is exposed to invasive tumor cell lines in the presence andabsence of PTA or PTA-derivatives. The invasion potential of the tumorcell is measured by the change in cell impedance as the HUVEC monolayerjunctions are compromised by the invading tumor cells. Cells can bemonitored over multiple days while the real-time impedance is recorded.

Since the number of cells that have been seeded is known, the CI isrelated to the quantitative measurement of the electrical impedancepresent in the well and it displays in plots the changes of cells thatadhere to the conducting metals on the bottom of the wells. Therefore,CI values increase or decrease in parallel with cell growth due to theinsulating properties of the cell membrane attached to the bottom of thewell. The data was processed according to the “sigmoidal dose-response”(variable slope nonlinear regression curve fit for an inhibitor), fromwhich the IC₅₀ was obtained. The software generated a standard errorvalue for the data points based on the fitted sigmoidal curve and thevariability of the points around that curve. As a curve type forcytotoxicity DRC (CI at a time point vs. concentration) was used.

Increased LC3-II levels are correlated with properties of metastasizingcancer cell lines. It is therefore thought that if LC3-II levels arereduced or inhibited, then the level of metastasizing cells shouldcorrespondingly decline. To test this hypothesis we have performedinvasion assays with two cancer cell lines (MCF-7, a breast cancer cellline and HepG2, a hepatocarcinoma cell line), the results are shown inFIG. 5.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method for treating cancer in a subject comprising administering tothe subject an effective amount of a compound of formula I:

or salt, or stereoisomer thereof, wherein R₁ is an, pyrimidyl,napthalenyl or heteronapthalenyl group which may be substituted with oneor more C₁-C₆ alkyl or substituted C₁-C₆ alkyl groups; wherein L is alinker group of 0 or 1, comprising an alkylamino group; wherein R₂ is anaryl, pyridyl or pyrimidyl group wherein the pyridyl or pyrimidyl groupis substituted at C₂-C₄ with one or more C₁-C₆ alkylamino C₁-C₆ alkyl,C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆alkylthio C₁-C₆ alkyl, C₆-C₁₄ arylthio, C₁-C₆ alkylsulfonyl C₁-C₆ alkyl,hydroxy C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₃-C₈cycloalkyl, C₁-C₆ alkylamino, di C₁-C₆ alkylamino, C₁-C₆ alkylthio,C₂-C₆ alkenylthio, C₂-C₆ alkynylthio, C₆-C₁₄ aryloxy, C₂-C₆ acyloxy,thio C₂-C₆ acyl, amido, and sulphonamido, and C₁-C₆ alkyl, and C₂-C₆alkenyl, C₂-C₆ alkynyl, and wherein the aryl group may be substituted atC₂-C₄ with one or more C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylaminoC₁-C₆ alkyl, C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ alkylthio C₁-C₆ alkyl,C₆-C₁₄ arylthio, C₁-C₆ alkylsulfonyl C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₃-C₈ cycloalkyl, heterocyclyl,C₁-C₆ alkylamino, di C₁-C₆ alkylamino, C₁-C₆ alkylthio, C₂-C₆alkenylthio, C₂-C₆ alkynylthio, C₆-C₁₄ aryloxy, C₂-C₆ acyloxy, thioC₂-C₆ acyl, amido, and sulphonamido, and C₁-C₆ alkyl, and C₂-C₆ alkenyl,C₂-C₆ alkynyl, or a halogen, wherein each of alkyl, aryl, orheterocyclyl moiety may be unsubstituted or substituted with one or moresubstituents selected from the group consisting of halo, hydroxy,carboxy, phosphoryl, phosphonyl, phosphono C₁-C₆ alkyl, carboxy C₁-C₆alkyl, dicarboxy C₁-C₆ alkyl, dicarboxy halo C₁-C₆ alkyl, sulfonyl,cyano, nitro, alkoxy, alkylthio, acyl, acyloxy, thioacyl, acylthio,aryloxy, amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino,guanidino, aldehydo, ureido, and aminocarbonyl; and optionally whereinR₂ is linked to the aminothiazole ring of formula I by an amide linkage,or a salt solvate, or stereoisomer thereof.
 2. The method of claim 1,wherein the compound of claim 1 has the formula:

wherein R₂ is an aryl which may be substituted at C₂-C₄ with one or moreC₁-C₆ alkyl groups or a halogen, or a or a pyrimidyl group which issubstituted at C₂-C₄ with one or more C₁-C₆ alkyl groups or a halogen,and R₃ is a C₁-C₆ alkyl or substituted C₁-C₆ alkyl group.
 3. The methodof claim 1, wherein the compound of claim 1 has the formula:

wherein R₂ is an aryl which may be substituted at C₂-C₄ with one or moreC₁-C₆ alkyl groups or a halogen, or a or a pyrimidyl group which issubstituted at C₂-C₄ with one or more C₁-C₆ alkyl groups or a halogen,and R₃ is a C₁-C₆ alkyl or substituted C₁-C₆ alkyl group.
 4. The methodof claim 1, wherein the compound of claim 1 is selected from the groupconsisting of:


5. The method of claim 1, wherein the composition further comprises atleast one chemotherapeutic agent.
 6. The method of claim 5, wherein thechemotherapeutic agents are selected from the group consisting ofalkylating agents, angiogenesis inhibitors, aromatase inhibitors,antimetabolites, anthracyclines, antitumor antibiotics, monoclonalantibodies, platinums, topoisomerase inhibitors, and plant alkaloids. 7.A method for treating cancer in a subject comprising administering tothe subject an effective amount of a pharmaceutical compositioncomprising a compound of formula I:

or salt, or stereoisomer thereof, wherein R₁ is an, pyrimidyl,napthalenyl or heteronapthalenyl group which may be substituted with oneor more C₁-C₆ alkyl or substituted C₁-C₆ alkyl groups; wherein L is alinker group of 0 or 1, comprising an alkylamino group; wherein R₂ is anaryl, pyridyl or pyrimidyl group wherein the pyridyl or pyrimidyl groupis substituted at C₂-C₄ with one or more C₁-C₆ alkylamino C₁-C₆ alkyl,C₁-C₆ dialkylamino C₁-C₆ alkyl, C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆alkylthio C₁-C₆ alkyl, C₆-C₁₄ arylthio, C₁-C₆ alkylsulfonyl C₁-C₆ alkyl,hydroxy C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₃-C₈cycloalkyl, C₁-C₆ alkylamino, di C₁-C₆ alkylamino, C₁-C₆ alkylthio,C₂-C₆ alkenylthio, C₂-C₆ alkynylthio, C₆-C₁₄ aryloxy, C₂-C₆ acyloxy,thio C₂-C₆ acyl, amido, and sulphonamido, and C₁-C₆ alkyl, and C₂-C₆alkenyl, C₂-C₆ alkynyl, and wherein the aryl group may be substituted atC₂-C₄ with one or more C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ dialkylaminoC₁-C₆ alkyl, C₁-C₆ alkylamino C₁-C₆ alkyl, C₁-C₆ alkylthio C₁-C₆ alkyl,C₆-C₁₄ arylthio, C₁-C₆ alkylsulfonyl C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ alkoxy C₁-C₆ alkyl, C₃-C₈ cycloalkyl, heterocyclyl,C₁-C₆ alkylamino, di C₁-C₆ alkylamino, C₁-C₆ alkylthio, C₂-C₆alkenylthio, C₂-C₆ alkynylthio, C₆-C₁₄ aryloxy, C₂-C₆ acyloxy, thioC₂-C₆ acyl, amido, and sulphonamido, and C₁-C₆ alkyl, and C₂-C₆ alkenyl,C₂-C₆ alkynyl, or a halogen, wherein each of alkyl, aryl, orheterocyclyl moiety may be unsubstituted or substituted with one or moresubstituents selected from the group consisting of halo, hydroxy,carboxy, phosphoryl, phosphonyl, phosphono C₁-C₆ alkyl, carboxy C₁-C₆alkyl, dicarboxy C₁-C₆ alkyl, dicarboxy halo C₁-C₆ alkyl, sulfonyl,cyano, nitro, alkoxy, alkylthio, acyl, acyloxy, thioacyl, acylthio,aryloxy, amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino,guanidino, aldehydo, ureido, and aminocarbonyl; and optionally whereinR₂ is linked to the aminothiazole ring of formula I by an amide linkage,or a salt solvate, or stereoisomer thereof, and a pharmaceuticallyacceptable carrier.
 8. The method of claim 7, wherein the compound ofthe pharmaceutical composition of claim 7 has the formula:

wherein R₂ is an aryl which may be substituted at C₂-C₄ with one or moreC₁-C₆ alkyl groups or a halogen, or a or a pyrimidyl group which issubstituted at C₂-C₄ with one or more C₁-C₆ alkyl groups or a halogen,and R₃ is a C₁-C₆ alkyl or substituted C₁-C₆ alkyl group.
 9. The methodof claim 7, wherein the compound of the pharmaceutical composition ofclaim 7 has the formula:

wherein R₂ is an aryl which may be substituted at C₂-C₄ with one or moreC₁-C₆ alkyl groups or a halogen, or a or a pyrimidyl group which issubstituted at C₂-C₄ with one or more C₁-C₆ alkyl groups or a halogen,and R₃ is a C₁-C₆ alkyl or substituted C₁-C₆ alkyl group.
 10. The methodof claim 7, wherein the compound of the pharmaceutical composition ofclaim 7 is selected from the group consisting of:


11. The method of claim 7, wherein the composition further comprises atleast one chemotherapeutic agent.
 12. The method of claim 11, whereinthe chemotherapeutic agents are selected from the group consisting ofalkylating agents, angiogenesis inhibitors, aromatase inhibitors,antimetabolites, anthracyclines, antitumor antibiotics, monoclonalantibodies, platinums, topoisomerase inhibitors, and plant alkaloids.